Figure 3.
Surplus EPO-dependent EPs undergo apoptosis in vivo. (A) Gating and representative flow cytometry data are shown for annexin V staining of Ter119+ EPs in marrow from TGFβ1FL/FL (top row) and TGFβ1ΔMk/ΔMk (bottom row) mice. Quantification of the number of marrow, annexin V staining, apoptotic Ter119+ erythroid cells is shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice (n = 12 per group). (B) Gating and representative flow cytometry data are shown spleen cells as described for panel A. Quantification of splenic apoptotic Ter119+ erythroid cells is shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice (n = 4-6 per group). (C) Flow cytometry gating strategy using Lin− erythroid LinE− (cells not expressing CD3, B220, Gr1, or CD11b), CD44, Ter119, Hoechst (DNA), and annexin V to characterize erythroid maturation. (D) Quantification of EPs is shown (n = 3 per group). Viable cells and annexin V+ apoptotic cells for TGFβ1FL/FL (gray/green) and TGFβ1ΔMk/ΔMk (black/blue) mice, respectively. (E) Representative flow cytometry showing staining of Kit and Epor within the annexin V+/Ter119+ population identifies excess Epor+/Kit− erythroblasts within the TGFβ1ΔMk/ΔMk marrow. (F) Plasma EPO levels are shown as assessed by enzyme-linked immunosorbent assay (ELISA; n = 8 per group). (G) TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice were treated with 300 U/kg Epo daily for 5 days and euthanized on day 6 for analysis. (H) Representative annexin V staining of marrow EPCs is shown after PBS (top) or Epo (bottom) treatment of TGFβ1FL/FL (left) and TGFβ1ΔMk/ΔMk (right) mice. The number of annexin V+ apoptotic Ter119+ EPCs is shown in marrow (I) and spleen (J) before and after EPO treatment (n = 4-12 per group). (K) Apoptosis within maturing EPs is shown for marrow cells after EPO treatment, as shown in panel D. Viable cells and annexin V+ apoptotic cells for Epo-treated TGFβ1FL/FL (pink/green) and TGFβ1ΔMk/ΔMk (red/blue) mice, respectively (n = 4 per group). Reticulocyte (Retic) counts (L) and RBCs (M) are shown for TGFβ1FL/FL (gray/pink) and TGFβ1ΔMk/ΔMk (black/red) mice before and after Epo (n = 6 per group). All of the quantified data are shown as mean plus or minus SD (*P < .05, **P < .01, ***P < .001, or if not shown, the comparison was not significant). AV, annexin V; CBC, complete blood count; Rx, prescription.

Surplus EPO-dependent EPs undergo apoptosis in vivo. (A) Gating and representative flow cytometry data are shown for annexin V staining of Ter119+ EPs in marrow from TGFβ1FL/FL (top row) and TGFβ1ΔMk/ΔMk (bottom row) mice. Quantification of the number of marrow, annexin V staining, apoptotic Ter119+ erythroid cells is shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice (n = 12 per group). (B) Gating and representative flow cytometry data are shown spleen cells as described for panel A. Quantification of splenic apoptotic Ter119+ erythroid cells is shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice (n = 4-6 per group). (C) Flow cytometry gating strategy using Lin− erythroid LinE− (cells not expressing CD3, B220, Gr1, or CD11b), CD44, Ter119, Hoechst (DNA), and annexin V to characterize erythroid maturation. (D) Quantification of EPs is shown (n = 3 per group). Viable cells and annexin V+ apoptotic cells for TGFβ1FL/FL (gray/green) and TGFβ1ΔMk/ΔMk (black/blue) mice, respectively. (E) Representative flow cytometry showing staining of Kit and Epor within the annexin V+/Ter119+ population identifies excess Epor+/Kit− erythroblasts within the TGFβ1ΔMk/ΔMk marrow. (F) Plasma EPO levels are shown as assessed by enzyme-linked immunosorbent assay (ELISA; n = 8 per group). (G) TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice were treated with 300 U/kg Epo daily for 5 days and euthanized on day 6 for analysis. (H) Representative annexin V staining of marrow EPCs is shown after PBS (top) or Epo (bottom) treatment of TGFβ1FL/FL (left) and TGFβ1ΔMk/ΔMk (right) mice. The number of annexin V+ apoptotic Ter119+ EPCs is shown in marrow (I) and spleen (J) before and after EPO treatment (n = 4-12 per group). (K) Apoptosis within maturing EPs is shown for marrow cells after EPO treatment, as shown in panel D. Viable cells and annexin V+ apoptotic cells for Epo-treated TGFβ1FL/FL (pink/green) and TGFβ1ΔMk/ΔMk (red/blue) mice, respectively (n = 4 per group). Reticulocyte (Retic) counts (L) and RBCs (M) are shown for TGFβ1FL/FL (gray/pink) and TGFβ1ΔMk/ΔMk (black/red) mice before and after Epo (n = 6 per group). All of the quantified data are shown as mean plus or minus SD (*P < .05, **P < .01, ***P < .001, or if not shown, the comparison was not significant). AV, annexin V; CBC, complete blood count; Rx, prescription.

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